We are trying to understand the central but poorly understood organelle that organizes the cytoplasm in eukaryotic cells. Our unique approach to the study of the centrosome emphasizes the dissection of the centrosome subunit as a multi-protein complex that binds to the minus end of a single microtubule. As our model system for such a study, we are using the early Drosophila embryo, whose centrosomal mechanisms can be safely assumed to be essentially identical to those of a human cell. Drosophila has the following advantages: large quantities of embryos are easily obtained; the syncytial early embryos are large, robust, single cells that are easily microinjected with proteins; these embryos have a highly organized cytoplasm, with easily observable, rapid changes in spatial organization caused by the centrosome; and powerful genetic analyses of centrosome function are possible. In the past grant period, we have discovered and extensively characterized three Drosophila centrosomal proteins (and cDNAs): DMAP60, DMAP190, and the l4OkD LK6 protein kinase, as well as partially characterizing others. In addition, we have complete cDNA clones for two proteins suspected to be intimately involved with the actin-induced membrane-furrowing that is caused by the centrosome: a 134kD actin-binding protein called ABP8, and the product of a maternal-effect mutant gene that we have named scrambled. The current proposal has four specific aims: 1) to scale up and refine our existing Drosophila embryo centrosome purification to obtain biochemical quantities of active centrosomes of sufficient purity to identify a full complement of centrosomal proteins; 2) to dissect the structure and function of a complex of three Drosophila centrosomal proteins studied in our laboratory (DMAP190, DMAP60, and gamma-tubulin) and determine the individual contributions of its components to the microtubule binding observed for the entire complex; 3) to use both biochemical and genetic approaches to decipher the role of the LK6 protein kinase in the regulation of centrosome function; and 4) to identify the function of the ABP8 and scrambled proteins in an attempt to decipher how actin filament-induced membrane furrowing is linked to the centrosome.